An SPR sensor and chip for detecting heavy metal ions using chitosan carbon dots

Abstract

The invention discloses an SPR sensor and a chip for detecting heavy metal ions by using chitosan carbon dots, and belongs to the cross-technical field of surface plasmon resonance (SPR) technology, polymer materials, and SPR active layer film preparation. The sensor comprises a glass prism and an SPR sensor chip which are arranged in sequence, and the SPR sensor chip is composed of a chromium film, a metal film, and an active layer film in sequence from top to bottom. The SPR sensor for detecting heavy metal ions by using chitosan carbon dots of the invention improves the characteristics of a traditional metal film, a chromium film is added to the total reflection surface of the prism before the metal film is plated, and a chitosan-carbon dot film is added to the surface of the metal film at the same time, and the strong adsorption effect of the chitosan carbon dots on 2,3-diaminophenazine is utilized to realize the highly sensitive and specific detection of heavy metal copper ions.

Description

Technical Field

[0001] The invention relates to the cross-technical field of surface plasmon resonance (SPR) technology, polymer materials and SPR active layer film preparation, and in particular to an SPR sensor and chip for detecting heavy metal ions using chitosan carbon dots. Background Art

[0002] Copper is an essential trace element in the human body. Insufficient intake will affect the normal physiological functions of the human body, and excessive intake will cause heavy metal poisoning, damage to the liver, kidneys and other organs, and cause some diseases, such as Wilson's disease. About 90% of copper ions in a normal human body are excreted through bile. When the human body ingests excessive copper ions, copper ions cannot be effectively excreted through bile, resulting in their accumulation in liver cells, damaging mitochondria and other cells through oxygen stress damage, and then causing fatty lesions, necrosis and apoptosis of liver cells. After liver cell destruction, free copper is released into the blood, which can cause damage to other organs. In addition, the half-life of ceruloplasmin that is not bound to copper ions is shortened in the blood, resulting in a decrease in blood ceruloplasmin in patients with Wilson's disease. For the sake of people's healthy life, trace detection of copper ions is particularly important. It is of great significance to carry out high-sensitivity and specific detection methods and sensing mechanism research of copper ions.

[0003] The drinking water standards in our country stipulate that the content of divalent copper ions in water should be less than 20μM, and the drinking water standards in the United States and the European Union stipulate that the content of divalent copper ions in water should be less than 20μM and 30μM respectively. Therefore, the detection of copper ions in food and the environment is one of the important means to ensure people's health. The detection of trace copper ions in food and the environment is of great significance, so people have carried out in-depth research on copper ion detection.

[0004] The main methods for detecting the content of heavy metal copper ions include atomic absorption spectroscopy, atomic emission spectroscopy, atomic fluorescence spectroscopy, mass spectrometry, enzyme inhibition and electrochemical analysis. These traditional instrumental analysis and testing methods have their own advantages, but they are cumbersome, time-consuming, and low in sensitivity. The specificity of detection needs to be further improved, and they cannot be applied to on-site analysis and testing, which has always been a problem in the current detection of heavy metal copper ions.

[0005] SPR sensing technology is an optical detection method. It was first proposed by Wood et al. in the early 20th century and was first used in the field of gas and biosensing in the 1980s. Compared with traditional detection methods, it has the advantages of no labeling, high sensitivity, fast response, and real-time monitoring. The typical model of prism-type SPR is a total reflection prism. The total reflection surface is coated with one or more metal films. When the incident angle of the light signal on the total reflection surface is greater than the critical angle, the light signal is totally reflected. Under this condition, the p-polarized light of the light signal transmits the light signal to the metal film medium at the interface between the total reflection surface of the prism and the metal film. The light signal is the evanescent wave, and its amplitude decays exponentially, causing the free electrons in the metal medium to form a surface plasma wave. If the incident angle or wavelength of the light signal is adjusted to a certain value, the frequency of the surface plasma wave is equal to the wave number of the evanescent wave, and they will undergo energy coupling and resonate. Then the energy of the incident light signal is absorbed, and the emission light intensity is reduced accordingly. At this time, a resonance peak appears in the reflection spectrum, and the corresponding incident angle is called the resonance angle. SPR sensing technology is divided into angle modulation, wavelength modulation and intensity modulation. With the unique advantages of SPR, SPR sensing technology is becoming more and more popular in various fields, and has great advantages in heavy metal ion and pesticide residue monitoring of food safety and environmental monitoring.

[0006] The existing prism SPR sensors have various structures and each has its own characteristics. Their common feature is to use a metal film (gold or silver film) to modify a thin film on the surface for highly selective and effective adsorption of one or several substances. However, the existing prism SPR sensors have many shortcomings, such as: some detection takes a long time, some modified films are not firm and easily swell in the solution, and some have many film layers and the preparation process is too complicated.

[0007] Chinese patent document number CN102621104A, published on August 1, 2012, discloses a D-type optical fiber sensor based on graphene film sensitization, the sensor includes a D-type optical fiber, a silver film layer on the polished surface of the D-type optical fiber, and a graphene film layer on the surface of the silver film layer. This method uses the deposition or growth of graphene film material on the surface of the silver film to increase the sensitivity of the SPR sensing structure, but the fixation stability and non-specific adsorption control of graphene film on biomolecules are not as mature as the preparation technology of gold and silver film surfaces.

[0008] Chinese patent document number CN109883999A, published on June 14, 2019, discloses a prism SPR heavy metal ion sensor for specific detection of copper ions. The invention adds a magnetic TiO2 film between the surface of the carrier substrate of the chip and its metal film; the other surface of the metal film is covered with a magnetic polyaniline nanoparticle-chitosan film. In this method, the magnetic polyaniline nanoparticle-chitosan modified film generates a magnetic attraction to improve the firmness of the modified film, but the magnetic polyaniline nanoparticles affect the control of uniformity due to their magnetism, and it is difficult to control the uniformity on the film, which causes the film to affect the performance of the sensor. In addition, the film structure is relatively complex, the preparation process is relatively cumbersome, and the process control requirements are high. Summary of the invention

[0009] The purpose of the present invention is to provide an SPR sensor and chip for detecting heavy metal ions using chitosan carbon dots, so as to solve the problems existing in the above-mentioned prior art.

[0010] To achieve the above object, the present invention provides the following solutions:

[0011] The invention provides an SPR sensor chip for detecting heavy metal ions by using chitosan carbon dots. A molecular sensitive film is added on the surface of a metal film, a chromium film is added on the other surface of the metal film, and the molecular sensitive film is an active layer film.

[0012] Furthermore, the metal film is a gold film or a silver film.

[0013] Silver film or gold film is used to generate SPR, and the sensitivity of the sensor chip in detecting heavy metal ions can be improved by optimizing the thickness of the silver film or gold film.

[0014] Furthermore, the thickness of the silver film is 25-45 nm; the thickness of the gold film is 10-20 nm.

[0015] Furthermore, the silver film and the gold film are coated by vacuum sputtering, magnetron sputtering or ion source assisted electron beam evaporation.

[0016] Furthermore, in a preferred embodiment of the present invention, the silver film is coated by vacuum sputtering, and the gold film is coated by magnetron sputtering.

[0017] Furthermore, the thickness of the chromium film is 3-5 nm.

[0018] Furthermore, the chromium film is deposited by pulse deposition.

[0019] Furthermore, the active layer membrane is a chitosan-carbon dot membrane, which is prepared by slowly sprinkling carbon dots into a prepared chitosan solution, ultrasonically stirring for 5-20 minutes, and coating by spin coating; wherein the volume mass ratio of the chitosan solution to the carbon dots is 30mL:0.5-10g; the thickness of the chitosan-carbon dot membrane is 30-50nm, and the chitosan-carbon dot membrane is coated by ion source assisted electron beam evaporation.

[0020] Furthermore, the chitosan solution is prepared by dissolving chitosan with a molecular weight of more than 300,000 and a deacetylation degree of more than 90% in a hydrochloric acid solution until the chitosan is completely dissolved, adding a genipin solution, and stirring until the chitosan solution is completely cross-linked.

[0021] Furthermore, the mass ratio of chitosan to genipin is 100:3-15; and the concentration of the hydrochloric acid solution is 0.2-2 mol / L.

[0022] Furthermore, the carbon dots are prepared by adding chitosan with a molecular weight of 50,000-400,000 and a deacetylation degree of 90-95%, diaminoethane and deionized water into a polytetrafluoroethylene reactor, reacting at 180-200° C. for 5-20 hours, cooling to room temperature, dialyzing with deionized water for 15-24 hours, freeze-drying to carbon dot powder, and storing at 4° C. for later use.

[0023] Furthermore, the mass ratio of the chitosan, diaminoethane and deionized water is 1-1.5:1:40-60.

[0024] The present invention also provides an SPR sensor for detecting heavy metal ions using chitosan carbon dots, comprising a glass prism and the above-mentioned SPR sensor chip which are arranged in sequence, wherein the glass prism is arranged on the surface of the chromium film.

[0025] Before using the SPR sensor of the present invention to detect heavy metal ions in a solution, a NaOH solution is dripped into the solution to be tested to adjust the pH value to 9-10, and an o-phenylenediamine solution is added at a volume ratio of 1:1 between the heavy metal ion solution to be tested and the o-phenylenediamine solution. If divalent copper ions exist in the solution to be tested, the o-phenylenediamine will be oxidized to 2,3-diaminophenazine by the divalent copper ions, and the chitosan carbon dots on the active layer film on the surface of the metal film of the SPR sensor have a strong adsorption effect on 2,3-diaminophenazine, thereby rapidly and highly sensitively changing the SPR spectral signal, thereby realizing specific detection of heavy metal copper ions.

[0026] The present invention discloses the following technical effects:

[0027] The carbon dot powder of the present invention does not have magnetism, so it does not affect the uniformity of the film, the preparation process is relatively simple and easy to implement, and the active layer film has better stability. In addition, the use of genipin crosslinking agent significantly reduces cytotoxicity compared to glutaraldehyde crosslinking agent, and the stability of the crosslinked product is better.

[0028] The SPR sensor for detecting heavy metal ions using chitosan carbon dots described in the present invention improves the characteristics of traditional metal films. Before plating the metal film, a chromium film is added to the total reflection surface of the prism, and a chitosan-carbon dot film is added to the surface of the metal film. The strong adsorption effect of chitosan carbon dots on 2,3-diaminophenazine is utilized to achieve high-sensitivity and high-specificity detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0030] Figure 1 Schematic diagram of SPR sensor for detecting heavy metal ions using chitosan carbon dots;

[0031] Among them, 1-total reflection prism for exciting SPR; 2-chromium film; 3-metal film; 4-active layer film. DETAILED DESCRIPTION

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0034] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0035] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0036] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0037] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0038] Example 1

[0039] like Figure 1 As shown, an SPR sensor for detecting heavy metal ions using chitosan carbon dots comprises a glass prism 1 and an SPR sensor chip which are arranged in sequence. The SPR sensor chip is composed of a chromium film 2, a gold film 3, and an active layer film 4 from top to bottom.

[0040] Among them, on the total reflection surface of the SPR glass prism 1, a chromium film 2 is prepared by pulse deposition technology, and the thickness of the chromium film 2 is 4nm; a gold film 3 with a thickness of 15nm is added on the surface of the chromium film 2 of the SPR glass prism 1 by vacuum sputtering method; and then an active layer film 4 with a thickness of 40nm is added on the surface of the gold film 3 by spin coating.

[0041] Wherein, the active layer membrane is a chitosan-carbon dot membrane, and its preparation method comprises the following steps:

[0042] (1) Preparation of carbon dots: chitosan with a molecular weight of 350,000 and a deacetylation degree of 95%, diaminoethane and deionized water were added into a polytetrafluoroethylene reactor in a mass ratio of 13:1:50, reacted at 190°C for 15 h, cooled to room temperature, dialyzed with deionized water for 20 h, freeze-dried to carbon dot powder, and stored at 4°C for later use.

[0043] (2) Preparation of chitosan solution: Dissolve 1.9 g of chitosan with a molecular weight of more than 300,000 and a deacetylation degree of more than 90% in a 1 mol / L hydrochloric acid solution until it is completely dissolved, then add the genipin solution in a ratio of chitosan to genipin of 100:9, and stir until the chitosan solution is completely cross-linked.

[0044] (3) Preparation of chitosan-carbon dot film: Take 30 mL of the chitosan solution prepared in step (2), slowly sprinkle 5 g of the carbon dots prepared in step (1) into the solution, and stir ultrasonically for 13 min. Then, use spin coating to prepare an active layer film with a large amount of chitosan carbon dots on the gold film surface of the total reflection surface of the SPR prism, thereby obtaining an SPR sensor using chitosan carbon dots to detect heavy metal ions.

[0045] Example 2

[0046] like Figure 1 As shown, an SPR sensor for detecting heavy metal ions using chitosan carbon dots comprises a glass prism 1 and an SPR sensor chip which are arranged in sequence. The SPR sensor chip is composed of a chromium film 2, a silver film 3, and an active layer film 4 from top to bottom.

[0047] Among them, on the total reflection surface of the SPR glass prism 1, a chromium film 2 is prepared by pulse deposition technology, and the thickness of the chromium film 2 is 3nm; a silver film 3 with a thickness of 35nm is added on the surface of the chromium film 2 of the SPR glass prism 1 by vacuum sputtering method; and then an active layer film 4 with a thickness of 40nm is added on the surface of the silver film 3 by spin coating.

[0048] Wherein, the active layer membrane is a chitosan-carbon dot membrane, and its preparation method comprises the following steps:

[0049] (1) Preparation of carbon dots: chitosan with a molecular weight of 350,000 and a deacetylation degree of 95%, diaminoethane and deionized water were added into a polytetrafluoroethylene reactor in a mass ratio of 13:1:50, reacted at 190°C for 15 h, cooled to room temperature, dialyzed with deionized water for 20 h, freeze-dried to carbon dot powder, and stored at 4°C for later use.

[0050] (2) Preparation of chitosan solution: Dissolve 1.9 g of chitosan with a molecular weight of more than 300,000 and a deacetylation degree of more than 90% in a 1 mol / L hydrochloric acid solution until it is completely dissolved, then add the genipin solution in a ratio of chitosan to genipin of 100:9, and stir until the chitosan solution is completely cross-linked.

[0051] (3) Preparation of chitosan-carbon dot film: Take 30 mL of the chitosan solution prepared in step (2), slowly sprinkle 5 g of the carbon dots prepared in step (1) into the solution, and stir ultrasonically for 13 min. Then, use spin coating to prepare an active layer film with a large amount of chitosan carbon dots on the silver film surface of the total reflection surface of the SPR prism, and obtain an SPR sensor using chitosan carbon dots to detect heavy metal ions.

[0052] Example 3

[0053] like Figure 1 As shown, an SPR sensor for detecting heavy metal ions using chitosan carbon dots comprises a glass prism 1 and an SPR sensor chip which are arranged in sequence. The SPR sensor chip is composed of a chromium film 2, a gold film 3, and an active layer film 4 from top to bottom.

[0054] Among them, on the total reflection surface of the SPR glass prism 1, a chromium film 2 is prepared by pulse deposition technology, and the thickness of the chromium film 2 is 2nm; a gold film with a thickness of 12nm is added on the surface of the chromium film 2 of the SPR glass prism 1 by vacuum sputtering method; and then an active layer film 4 with a thickness of 45nm is added on the surface of the gold film 3 by spin coating.

[0055] Wherein, the active layer membrane is a chitosan-carbon dot membrane, and its preparation method comprises the following steps:

[0056] (1) Preparation of carbon dots: chitosan with a molecular weight of 100,000 and a deacetylation degree of 91%, diaminoethane and deionized water were added into a polytetrafluoroethylene reactor in a mass ratio of 14:1:45, reacted at 185°C for 18 h, cooled to room temperature, dialyzed with deionized water for 18 h, freeze-dried to carbon dot powder, and stored at 4°C for later use.

[0057] (2) Preparation of chitosan solution: Dissolve 2.5 g of chitosan with a molecular weight of more than 300,000 and a degree of deacetylation of more than 90% in a 0.5 mol / L hydrochloric acid solution until it is completely dissolved, then add the genipin solution in a ratio of chitosan to genipin of 100:5, and stir until the chitosan solution is completely cross-linked.

[0058] (3) Preparation of chitosan-carbon dot film: Take 30 mL of the chitosan solution prepared in step (2), slowly sprinkle 8 g of the carbon dots prepared in step (1) into the solution, and stir ultrasonically for 7 minutes. Then, use spin coating to prepare an active layer film with a large number of chitosan carbon dots on the gold film surface of the total reflection surface of the SPR prism, and obtain an SPR sensor using chitosan carbon dots to detect heavy metal ions.

[0059] Example 4

[0060] like Figure 1As shown, an SPR sensor for detecting heavy metal ions using chitosan carbon dots comprises a glass prism 1 and an SPR sensor chip which are arranged in sequence. The SPR sensor chip is composed of a chromium film 2, a gold film 3, and an active layer film 4 from top to bottom.

[0061] Among them, on the total reflection surface of the SPR glass prism 1, a chromium film 2 is prepared by pulse deposition technology, and the thickness of the chromium film 2 is 5nm; a gold film 3 with a thickness of 18nm is added on the surface of the chromium film 2 of the SPR glass prism 1 by vacuum sputtering method; and then an active layer film 4 with a thickness of 35nm is added on the surface of the gold film 3 by spin coating.

[0062] Wherein, the active layer membrane is a chitosan-carbon dot membrane, and its preparation method comprises the following steps:

[0063] (1) Preparation of carbon dots: chitosan with a molecular weight of 300,000 and a degree of deacetylation of 94%, diaminoethane and deionized water were added into a polytetrafluoroethylene reactor at a mass ratio of 1.1:1:55, reacted at 195°C for 8 h, cooled to room temperature, dialyzed with deionized water for 23 h, freeze-dried to carbon dot powder, and stored at 4°C for later use.

[0064] (2) Preparation of chitosan solution: Dissolve 0.8 g of chitosan with a molecular weight of more than 300,000 and a degree of deacetylation of more than 90% in a 1.5 mol / L hydrochloric acid solution until it is completely dissolved, then add genipin solution in a ratio of chitosan to genipin of 100:14, and stir until the chitosan solution is completely cross-linked.

[0065] (3) Preparation of chitosan-carbon dot film: Take 30 mL of the chitosan solution prepared in step (2), slowly sprinkle 0.9 g of the carbon dots prepared in step (1) into the solution, and stir ultrasonically for 18 minutes. Then, use spin coating to prepare an active layer film with a large amount of chitosan carbon dots on the gold film surface of the total reflection surface of the SPR prism, and obtain an SPR sensor that uses chitosan carbon dots to detect heavy metal ions.

[0066] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. An SPR sensor chip for detecting heavy metal ions using chitosan carbon dots, characterized in that: A molecular sensitive film is added to the surface of the metal film, and a chromium film is added to the other surface of the metal film, wherein the molecular sensitive film is an active layer film; The active layer membrane is a chitosan-carbon dot membrane, wherein the chitosan-carbon dot membrane is prepared by slowly sprinkling carbon dots into a prepared chitosan solution, ultrasonically stirring for 5-20 minutes, and coating the membrane by spin coating; wherein the volume mass ratio of the chitosan solution to the carbon dots is 30 mL: 0.5-10 g; and the thickness of the chitosan-carbon dot membrane is 30-50 nm; The carbon dots are prepared by adding chitosan with a molecular weight of 50,000 to 400,000 and a deacetylation degree of 90 to 95%, diaminoethane and deionized water into a polytetrafluoroethylene reactor, reacting at 180 to 200° C. for 5 to 20 hours, cooling to room temperature, dialyzing with deionized water for 15 to 24 hours, freeze-drying to carbon dot powder, and storing at 4° C. for later use; The heavy metal ion is a divalent copper ion; Before detecting heavy metal ions in the solution, NaOH solution is dripped into the solution to be tested to adjust the pH value to 9-10, and o-phenylenediamine solution is added at a volume ratio of 1:1 between the heavy metal ion solution to be tested and the o-phenylenediamine solution. If divalent copper ions exist in the solution to be tested, the o-phenylenediamine will be oxidized to 2,3-diaminophenazine by the divalent copper ions, and the chitosan carbon dots on the active layer film on the surface of the metal film of the SPR sensor have a strong adsorption effect on 2,3-diaminophenazine, thereby changing the SPR spectral signal.

2. The SPR sensor chip for detecting heavy metal ions using chitosan carbon dots according to claim 1, characterized in that: The metal film is a gold film or a silver film.

3. The SPR sensor chip for detecting heavy metal ions using chitosan carbon dots according to claim 2, characterized in that: The thickness of the silver film is 25-45 nm; the thickness of the gold film is 10-20 nm.

4. The SPR sensor chip for detecting heavy metal ions using chitosan carbon dots according to claim 1, characterized in that: The thickness of the chromium film is 3-5 nm.

5. The SPR sensor chip for detecting heavy metal ions using chitosan carbon dots according to claim 1, characterized in that: The chitosan solution is prepared by dissolving chitosan with a molecular weight of more than 300,000 and a deacetylation degree of more than 90% in a hydrochloric acid solution until the chitosan is completely dissolved, adding a genipin solution, and stirring until the chitosan solution is completely cross-linked.

6. The SPR sensor chip for detecting heavy metal ions using chitosan carbon dots according to claim 5, characterized in that: The mass ratio of chitosan to genipin is 100:3-15; the concentration of the hydrochloric acid solution is 0.2-2 mol / L.

7. The SPR sensor chip for detecting heavy metal ions using chitosan carbon dots according to claim 1, characterized in that: The mass ratio of the chitosan, diaminoethane and deionized water is 1-1.5:1:40-60.

8. An SPR sensor for detecting heavy metal ions using chitosan carbon dots, characterized in that: The invention comprises a glass prism and the SPR sensor chip according to any one of claims 1 to 7 which are arranged in sequence, wherein the glass prism is arranged on the surface of the chromium film.

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